Glutamate‐286 mutants of cytochrome bo‐type ubiquinol oxidase from Escherichia coli: influence of mutations on the binuclear center structure revealed by FT‐IR and EPR spectroscopies¹

Abstract

Glutamate-286 mutants of cytochrome Ao-type ubiquinol oxidase from Escherichia coli were examined by EPR and FT-IR spectroscopies. We confirmed a very low enzymatic activity for E286Q. However, E286D retained one-third of the wild-type activity, probably due to the presence of the carboxylic group on the side-chain. The effect of the mutations at position 286 on the binuclear site was observed clearly in the EPR spectral change for the air-oxidized state. The effect was more significantly manifested in the presence of cyanide or azide in the oxidized state. In contrast, the mutations only slightly perturbed the binuclear center of the СО-reduced enzymes. These results indicate the importance of a direct through-bond connectivity between Cu B and Glu 286 via Pro 285 and His 284 .

Transient Binding of CO to CuB in Cytochrome c Oxidase Is Dynamically Linked to Structural Changes around a Carboxyl Group: A Time-Resolved Step-Scan Fourier Transform Infrared Investigation

“…Earlier studies have shown that in the E. coli ubiquinol oxidase cytochrome bo 3 , binding of CO to Cu B results in changes of the protein environment of E(I-286), but presumably no changes in the protonation state of the amino acid side chain (Puustinen et al, 1997). A connectivity between the binuclear center and E(I-286) was also found from EPR and FTIR studies of the ligand-binding induced changes in wild-type and EQ(I-286)/ED(I-286) mutant enzymes from the E. coli enzyme (Tsubaki et al, 1997).…”

Volume Changes Associated with CO Photolysis from Fully Reduced Bovine Heart Cytochrome aa3

“…Coupling between ligand binding to the binuclear center and proton translocation has recently been suggested. Studies utilizing FTIR spectroscopy have shown that CO binding to the Cu B site of E. coli cytochrome bo 3 perturbs Glu-286, which is located at the end of a putative proton channel (the “D” channel). − Site-directed mutagenisis of this residue in either E. coli or R. spheroidies results in diminished activity and no proton pumping activity. Interestingly, Glu-286 is not directly linked to Cu B or to any ligands of Cu B .…”

Electron Transfer Processes in Subunit I Mutants of CytochromeboQuinol Oxidase inEscherichia coli

“…26) Among the subunit I mutants examined in this study, only Glu286Asp retained partial activity (31% of the wild-type enzyme). 27) Upon pulse radiolysis, we found that a reduction of hemes occurred in two phases in all the mutants (Fig. 2, Table 1).…”